Cyclic peptide tube

ABSTRACT

Cyclic homodetic peptides having a repeating D-L-chirality motif are shown to have a stable disk conformation with the amino acid side chains extending radially outward and the carbonyl and amino groups extending axially upward or downward. Such cyclic peptides can be employed as subunits in the assembly of molecular tubes. Cyclic peptides having a repeating D-L-chirality motif and lacking mutually repulsive side-chains are shown to stack atop one another in an anti-parallel fashion and are shown to be held together by the formation of β-sheet hydrogen bonding. The stacked cyclic peptides form a molecular tube having a central channel. The diameter of the channel is determined by the size cyclic peptide. If the cyclic peptide includes ionizable amino acid residues, e.g. glutamic acid or lysine, assembly and disassembly of the molecular tubes can be controlled by varying the pH. If the cyclic peptide includes hydrophobic amino acid residues, the molecular tube will insert into a lipid membrane. In such instances, the molecular tube provides a transmembrane channel. The channel can be gated or ungated. Molecular tubes can be terminated with a terminal cyclic peptide having methylated amino groups in one orientation. Molecular tubes may be employed as drug carriers, molecular sieves, reaction vessels, membrane channels, and other uses.

This invention was made with government support under Contract No.N00014-94-1-0365 by the Office of Naval Research. The government hascertain rights in the invention.

FIELD OF INVENTION

The invention relates to cyclic peptides and to molecular tubestructures constructed from cyclic peptides. More particularly, theinvention relates to the use of cyclic peptides having amino acidsequences with a repeating D-L-chirality motif employable forconstructing self-assembling molecular tubes.

BACKGROUND

Cyclic peptides form a large class of natural and synthetic compounds.Naturally occurring cyclic peptides have diverse biological activities,e.g., antibiotics, toxins, hormones, and ion transport regulators.Naturally occurring cyclic peptides are not known to be synthesized viamRNA transcription, i.e., the amino acid sequence of naturally occurringcyclic peptides is not coded by the genome of the organism producing thematerial. Instead, the synthesis of naturally occurring cyclic peptidesis dependent upon a series of non-transcriptional enzymes specificallydedicated to the synthesis of these products. Many cyclic peptidesemploy both amide and non-amide linkages and incorporate unnatural aminoacids, i.e., amino acids not utilized in the mRNA transcriptionalsynthesis of linear proteins. Both D- and L-enantiomers of amino acidsare widely employed in natural and synthetic amino acids syntheticanalogs of several naturally occurring cyclic peptides have beendesigned and synthesized with modified biological activity.

Chemically, cyclic peptides are divided into two categories, i.e.,homodetic peptides and heterodetic peptides. Homodetic peptides consistentirely of amino acid residues linked to one another by amide bonds.The present application is directed entirely to cyclic homodeticpeptides. Heterodetic peptides include linkages other than amidelinkages, e.g., disulfide linkages and ester linkages. Depsipeptides area type of heterodetic peptide. Depsipeptides employ ester linkages.Valinomycin is a cyclic depsipeptide with an alternating chiralD-D-L-L-motif employing ester linkages within the ring. The presentapplication specifically excludes heterodetic peptides. The chemistry ofboth homodetic and heterodetic cyclic peptides is extensively reviewedby Ovchinnikov et al. (1992), The Proteins, Vol. V: 307-642.

Molecular tubes are not previously known to be formed by cyclic peptidesbut are known to be formed by linear peptides. For example, gramicidin Ais a linear pentadecapeptide having an alternating chiral D-L-motif.When integrated into a target bio-membrane, gramicidin A forms aleft-handed anti-parallel double-stranded helix with 5.6-6.4 amino acidresidues per turn. Gramicidin has an average outer diameter ofapproximately 16 Å and an average inner diameter of approximately 4.8 Å.The inner channel of gramicidin serves as a path for passivetransmembrane ion transport. (See: Wallace, B. A. et al. (1988) Science,44: 182-187; and Lang, D. (1988) Science, 44: 188-191.)

Molecular tubes may be formed from materials other that amino acids.Carbon tubes are disclosed by Iijima (Nature (1991), 354: 56-58) andEbbesen et al. (Nature (1992), 358, 220-222). These carbon tubes arecomposed of graphite and have a concentric close ended structure.Inorganic tubes find wide application in chemistry, e.g., micro- andmeso-porous inorganic solids known as zeolites are employed forenhancing a variety of reactions. The area of zeolites is reviewed byMeier et al., Atlas of Zeolite Structure Types, 2nd Edn (Butterworths,London, 1988).

What is needed is a method for assembling and disassembling moleculartubes of varying length and width using interchangeable subunits. Whatis needed is a versatile subunit for implementing the above method,i.e., a subunit which responds to a undergoes self-assembly andself-disassembly upon. What is needed is homodetic cyclic peptides whichcan be employed as subunits for self assembling and disassemblingmolecular tubes.

SUMMARY OF THE INVENTION

The invention includes cyclic homodetic peptides employable forassembling and disassembling molecular tubes, molecular tubes assembledfrom such cyclic homodetic peptides, and methods for assembling anddisassembling such molecular tubes.

Cyclic homodetic peptides included within the invention have a stabledisk conformation which facilitates the self-assembly of such peptidesto form molecular tubes. A stable disk conformation is achieved bydesigning the cyclic peptides with a repeating D-L-chirality motif.Conformance with this repeating chirality motif necessitates that theamino acid sequence of the cyclic peptide include only an even number ofamino acid residues. Since glycine lack chirality, conformance with thisrepeating chirality motif also necessitates that the amino acid sequenceof the cyclic peptide exclude glycine or minimize the inclusion ofglycine.

A stable disk conformation is further favored by limiting the size ofthe cyclic peptide, viz., the amino acid sequence of the cyclic peptideincludes between 6 and 16 amino acid residues total. The stability ofthe disk conformation of cyclic peptides tends to decline withincreasing ring size due to statistical mechanics considerations. Cyclicpeptides with ring sizes greater than 16 residues are less preferred dueto the low stability of their disk conformation.

Molecular tubes are assembled by stacking cyclic peptides atop oneanother. The resulting structure defines an interior channel. Thediameter of the interior channel is determined by the size of the cyclicpeptide, i.e., channel size increase with the size of the cyclicpeptide. Cyclic peptides having only 6 amino acid residues have a verysmall channel suitable for the passage or inclusion of small ions only;cyclic peptides having 16 amino acid residues have a very large channelsuitable for the passage or inclusion of small molecules; cyclicpeptides having 16 amino acid residues have a very large channelsuitable for the passage or inclusion of DNA or RNA.

The repeating D-L-chirality motif is thought to stabilize the diskconformation of cyclic homodetic peptides by lowering the energy of theoutwardly oriented conformation of amino acid side chain grouts. In theoutwardly oriented conformation, side chain groups of amino acidresidues are oriented perpendicular to the axis of the disk in aradially outward direction orienting the amino acid side chains in thisconformation also orients the backbone carboxyl groups and backboneamino hydrogens in a generally axial direction. Orienting the backbonecarboxyl groups and backbone amino hydrogens in this axial directionpredisposes cyclic peptides to stack atop one another in ananti-parallel fashion so as to form β-sheet hydrogen bonding.

The kinetics of assembly and disassembly cyclic peptide to formmolecular tubes can be controlled by the selection of amino acid sidechain groups. Cyclic peptides with ionizable amino acid side chainsdisplay pH dependent kinetics. Charged cyclic peptides are found toresist tube assembly; neutralized cyclic peptides are found to promotetube assembly. For example, cyclic peptides incorporating glutamic acidare found to spontaneously assemble into molecular tubes at acidic pHbut resist assembly into molecular tubes at alkaline pH. Pre-assembledmolecular tubes are found to spontaneously disassemble when the pH israised from acid pH to alkaline pH. Judicious selection of amino acidside chains can promote packing or aggregation of molecular tubes toform tubular bundles.

Cyclic peptides composed entirely or largely of hydrophobic amino acidresidues form molecular tubes within lipid bilayers. Such moleculartubes can span a membrane and provide an ion or molecular channel acrosssuch membrane. Such molecular tubes can be employed for loading cells orlipid vesicles with ions or molecules from the extra-vesicular space,depending upon the channel size of the tube. Transmembrane moleculartubes may also be gated so as to control the diffusion of ions andmolecules through the channel.

Molecular tubes may be loaded with ionic or molecular inclusions withinthe channel space. If such tubes are loaded with a drug, the tubes maybe employed as a drug delivery system. Release of the drug frommolecular tubes may occur by diffusion or by tubular disassembly.

Molecular tubes may also be employed to facilitate the controlled growthof inorganic clusters, semiconductors, and atomic scale wires by meansof tube assembly and/or diffusion within the tube channel to producematerials having novel optical and electronic properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a self-assembled molecular tube.Backbone-backbone hydrogen bonding interactions are shown but amino acidside chains are omitted for clarity. The internal diameter of thechannel is determined by the ring size of the cyclic peptide subunits.

FIG. 2 illustrates a molecular tube having eight cyclic peptides of thetype having eight hydrophobic amino acids each, such as cyclic peptides5 and 6. The molecular tube traverses a lipid bilayer to provide achannel, indicated by arrows, across the lipid bilayer.

FIGS. 3A-C compare the channel size of three different cyclic peptidesemployable for constructing transmembrane molecular tubes. D and Ldenote the chirality of the amino acid residues. R₁ and R₂ are sidechains of hydrophobic amino acid residues such as Val, Leu, Ile, Phe,and Trp.

FIG. 3A illustrates a cyclic hexa-peptide having a channel diameter ofapproximately 7 Å.

FIG. 3B illustrates a cyclic octa-peptide having a channel diameter ofapproximately 10 Å.

FIG. 3C illustrates a cyclic dodeca-peptide having a channel diameter ofapproximately 15 Å.

FIG. 4 illustrates a side view of a cap subunit, such as cyclic peptide7 and its interactions with an adjacent cyclic peptide subunit and thesurrounding media.

FIG. 5 illustrates a schematic representation of a molecular tube havinga gated channel.

FIG. 6 illustrates a schematic representation of a bidentatate (top) anda tetradentate gated channel.

FIG. 7 illustrates preferred ligands for use in the construction ofion-gated membrane channels and their anticipated metal ion selectivity.

FIG. 8 illustrates a membrane channel closed with respect to moleculartransport mediated by [M(Im)₄]^(B+) complex formation.

FIG. 9A illustrates the structure of cyclic peptide 1, i.e., an eightamino acid cyclic peptide having the amino acid sequencecyclo[-(Gln-D-Ala-Glu-D-Ala)₂-]. (Sequence No.: 1)

FIG. 9B illustrates a fragment of a molecular tube having four cyclicpeptides of type 1.

FIG. 10 illustrates the structure of cyclic peptide 2, i.e., an eightamino acid cyclic peptide having the amino acid sequencecyclo[-(Gln-D-Ala)₄-]. (Sequence No.: 2)

FIG. 11 illustrates the structure of cyclic peptide 3, i.e., an eightamino acid cyclic peptide having the amino acid sequencecyclo[-(Gln-D-Leu)₄-]. (Sequence No.: 3)

FIG. 12 illustrates the structure of cyclic peptide 4, i.e., an eightamino acid cyclic peptide having the amino acid sequencecyclo[-(Gln-D-Val)₄-]. (Sequence No.: 4)

FIG. 13 illustrates the structure of cyclic peptide 5, i.e., an eightamino acid cyclic peptide having the amino acid sequencecyclo[-(Phe-D-Leu)₄]. (Sequence No.: 5)

FIG. 14 illustrates the structure of cyclic peptide 6, i.e., an eightamino acid cyclic peptide having the amino acid sequencecyclo[-(Phe-D-Ala)₄-]. (Sequence No.: 6)

FIG. 15 illustrates the structure of cyclic peptide 7, i.e., an eightamino acid cyclic peptide having the amino acid sequencecyclo[-(Phe-N(Me)Ala₄-]. (Sequence No.: 7) Cyclic peptide 7 is aterminal cyclic peptide, i.e., it is capable of hydrogen bonding only onone side of the disk and therefore terminates the assembly of themolecular tube.

FIG. 16A illustrates the structure of cyclic peptide 8, i.e., a twelveamino acid cyclic peptide having the amino acid sequencecyclo[-(Gln-D-Ala-Glu-D-Ala)₃-]. (Sequence No.: 8)

FIG. 16B illustrates a fragment of a molecular tube having four cyclicpeptides of type 8.

FIG. 17 illustrates the hydrogen bonding of molecular tubes constructedwith cyclic peptide 1. At high pH, unfavorable electrostaticinteractions and high water solubility disfavors intermolecularinteractions. At low pH, specific intermolecular backbone-backbone andside chain-side chain hydrogen bonding interactions as well as the lowerwater solubility of the peptide monomers promotes the formation ofmolecular tube ensembles.

FIGS. 18A-E are micrographic images by electron microscopy and electrondiffraction of cyclic peptide tubes.

FIG. 18A is a low magnification image of a suspension of cyclic peptidetube particles, i.e., tube aggregates, adsorbed to carbon support film(scale bar=1 micron).

FIG. 18B is a low dose image of a frozen hydrated single particle ofcyclic peptide tubes. The particle measures ˜86×1180 nm.

FIG. 18C is an enlarged image of the boxed region in FIG. 18Billustrating longitudinal striations with a resolution of approximately˜10 Å.

FIG. 18D is an image enhancement of FIG. 18 C illustrating 14.9 Ålongitudinal striations representing side by side packing of cyclicpeptide tubes in the particle (scale bar=10 nm).

FIG. 18E represents an electron diffraction pattern recorded from asingle particle of cyclic peptide tubes showing orders of a 14.92 Åmeridional spacing and a 4.73 Å axial spacing. Axially the patternextends weakly to the third order (1.57 Å, data not shown) demonstratingthat the particles are highly ordered and crystalline.

FIGS. 19A-C illustrate infrared spectra (at 8 cm⁻¹ resolution) ofamide-I region.

FIG. 19A are the infrared spectra of monomeric peptide subunit in D₂O(4×10⁻³ M, pD=10, examined by attenuated total reflectance.

FIG. 19B is the infrared spectrum of particles of assembled cyclicpeptide tubes (KBr pellet).

FIG. 19C is the FT-IR spectrum of N—H stretch region of particles ofcyclic peptide tubes (KBr pellet). Components peaks are obtained bydeconvolution of the original spectrum with single component, mixedLorentzian and Gaussian functions using an iterative, linear leastsquares algorithm (“FIT”, Galactic Industries Corp.).

FIGS. 20A and B illustrate a three dimensional model of theself-assembled organic nanotubes formed by controlled acidification ofcyclo[-(D-Ala-Glu-D-Ala-Gln)₂-] (Sequence No.: 1) into rod-shapedcrystals, as illustrated in FIGS. 18A and 18B. View of the crystalpacking along the a axis is shown in FIG. 20 A. View of the crystalpacking along the c axis (bottom) is shown in FIG. 20B. The unit cell isindicated by the solid lines and the local two fold axis by theasterisk. The unit cell has dimensions a=9.5 Å, b=c=15.1 Å, and α=90,β=γ=99 degrees. The tube axis is along a (x).

FIGS. 21A and B illustrate a three dimensional model of theself-assembled nanotubes formed by controlled acidification ofcyclo[-(Gln-D-Ala-GluD-Ala)₃-] (Sequence No.: 8) into rod-shapedcrystals, as illustrated in FIGS. 23. View of the crystal packing isshown along the a-axis (for clarity, each nanotube is represented onlyby the local 2-fold dimer) in FIG. 21A. View of the crystal packing isshown along the c-axis in FIG. 21 B. The unit cell is indicated by thedashed lines and the local 2-fold axis by the asterisk. The unit cellhas the dimensions a=9.6 Å, b=c=25.66 Å, and α=120, β=γ=99 degrees. Thepositions of the side chains in this model are arbitrary. The tube axisis along a (x).

FIG. 22 illustrates a schematic representation of the strategy employedin the construction of self-assembled nanotubes of FIG. 21.Appropriately designed cyclic peptide subunits, under suitableconditions, stack to form hydrogen-bonded tubular structures (forclarity only backbone structure is represented). The ring size of thesubunit sets the internal diameter of the tubular ensemble. The chemicalstructure of the subunit is shown on the top right (D or L refers to theamino acid chirality).

FIGS. 23A-B illustrates, on the left, a low magnification electronmicrograph of nanotube suspensions of FIG. 21 adsorbed on carbon supportfilm (Scale bar, 1 mm) and, on the right, low-dose image of a frozenhydrated single nanotube particle. Longitudinal striations which areapproximately 25 Å apart are due to the side-by-side packing ofnanotubes.

FIGS. 24A and B illustrate infrared spectra of a membrane channelstructure formed by cyclo[-(Trp-D-Leu)₃-Gln-D-Leu-] (Sequence No.: 9).Figure A illustrates an infrared spectrum at 8-cm⁻¹ resolution of theamide-I region of the membrane channel structure. Figure B illustratesan infrared spectrum at 8-cm⁻¹ resolution of the N—H stretch region ofthe membrane channel structure. IR samples were prepared by the additionof a DMSO solution of the peptide to purified liposomes (20 to 50phospholipids per peptide subunit) followed by gel filtration on aSephadex G-25 column (for the method used to prepare the liposomes seelegend to FIG. 3). Liposomes were then applied to a CaF₂ disk and driedin vacuo for 30 minutes. Characteristic absorbances due to the peptidechannel ensemble are indicated in the figure.

FIG. 25 illustrates the ensuing proton efflux upon the addition of thechannel forming compounds indicated in FIG. 24. The proton efflux isexpressed in terms of the fractional change in the fluorescenceintensity of vesicle entrapped 5(6)-carboxyfluorescein as a function oftime (sampling time was at 0.3 seconds interval). Equal molar amounts ofchannel forming compounds were used in each experiment to allow directcomparisons of channel-mediated proton transport activity. The amount ofadded channel forming compounds in each experiment ranged from of 2 to20 nmoles corresponding to approximately 150 channels per liposome forthe lowest concentrations to 1100 channels per liposome for the highestamount of added compounds.

FIG. 26 illustrates a 140 second continuous K⁺ single channelconductance recording at 50 mV of the channel forming compoundsindicated in FIG. 24. Open-closed transitions indicate gating mechanismswhich may reflect structural flexibility or rapid assembly-disassemblyof the tubular membrane channel structures. Short and long channel lifetimes in the open state could be distinguished with values oft_((short))=0.7±0.03 ms and t_((long))=37.14±8.99 ms (total number ofevents 510) with an overall open probability P(O) of 0.29. Peptideconcentration at the subphase was 1.0×10⁻⁷ M. As is expected for anysuch self-assembling channel forming species, channel opening lifetimesshow significant dependence on the peptide concentration. Raising theadded peptide concentration in the subphase to 2.0×10⁻⁵ M, produces openchannel lifetimes of >30 open channel lifetimes of >30 seconds.

FIG. 27B illustrates a schematic representation of the self-assembledtubular transmembrane channel structure embedded in a lipid bilayermembrane emphasizing the antiparallel ring stacking, the presence ofextensive inter-subunit hydrogen-bonding interactions, and sidechain-lipid interactions (for clarity most side chains are omitted).FIG. 27A, the chemical structure of the peptide subunit, i.e.,cyclo[-Gln-(D-Leu-Trp)₄-D-Leu-] (Sequence No.: 9), is shown on the top(D- or L- refers to the amino acid chirality).

FIG. 28 illustrates a schematic illustration of channel-mediated glucosetransport and the enzyme coupled assay used to monitor the transportactivity with respect to the channel structure indicated in FIGS. 27A-B.Formation of a transmembrane pore structure(s) initiates glucose effluxfrom the liposome which can be directly monitored by measuring the rateof NADPH production. The enzymes and cofactors employed are hydrophilicand thus can not pass through the lipid membrane and are too large topenetrate the channel structure. Therefore, only the released glucosecan undergo the enzymatic reaction.

FIG. 29 illustrates the ensuing glucose efflux on the addition ofvarious amounts of the channel forming peptide of FIGS. 27A-B, viz.,open circles represent 15.0×10⁻⁶ M; closed circles represent 11.0×10⁻⁶M; open triangles represent 7.5×10⁻⁶ M; closed triangles represent5.6×10⁻⁶ M; and open diamonds represent 3.8×10⁻⁶ M. Efflux is expressedin terms of the amount of glucose released as a function of time(sampled at 90 sec intervals). The large unilamellar vesicles used inthis study contained 200 mM D-glucose. All curves are backgroundcorrected to remove any contribution from the nonspecific glucoseleakage from the liposomes.

DETAILED DESCRIPTION

Self-Assembled Nanotube with a 7 to 8 Å Pore:

The design, synthesis, and characterization of a molecular tube, i.e., acyclic peptide tube or “organic nanotube,” is described. A rationallydesigned 24-membered ring peptide structure can be constructed and shownto undergo a proton-triggered self-assembly to produce tubularstructures hundreds of nanometers long, with internal van der Waalsdiameter of 7 to 8 Å. Formation of tubular structures is established byElectron Microscopy (EM), electron diffraction studies, and FourierTransformed Infrared Spectroscopy (FT-IR). A three-dimensional model ofthe molecular tube consistent with the experimental observations is alsopresented. As opposed to the recently identified closed concentricgraphite tubes, cyclic peptide tubes, by virtue of the cyclic nature oftheir components, are open ended structures and have uniform shape andinternal diameter, e.g., Tsang, S. C. et al. (1993), Nature 362, 520-522and Ajayan, P. M. et al. (1993), Nature 362, 522-525 (1993). Theproton-triggered self-assembly process described herein is a highlyconvergent approach in which numerous ring-shaped peptide subunitsinteract through an extensive network of hydrogen bonds to formmolecular tube structures. Example I features an eight-residue cyclicpeptide with the following sequence: cyclo[-(D-Ala-Glu-D-Ala-Gln)₂-].(Sequence No.: 1) In designing the subunit, it is shown that cyclicpeptides with an even number of alternating D- and L-amino acids canadopt or sample a low energy ring-shaped flat conformation in which allbackbone amide functionalities lie approximately perpendicular to theplane of the structure. In this conformation, subunits can stack in ananti-parallel fashion and participate in backbone-backboneintermolecular hydrogen bonding to furnish a contiguous β-sheetstructure. Moreover, because of the alternating D- and L-amino acidsequence, peptide side chains must \necessarily lie on the exterior ofthe ensemble thereby creating the desired hollow tubular core structure.In the present example, the ionization state of the glutamic acid sidechain functionality is exploited as the trigger mechanism for theinitiation of the self-assembly and self-disassembly processed inaqueous solutions. At alkaline pH, the large repulsive intermolecularelectrostatic interactions between the negatively charged carboxylateside chains disfavors ring stacking and at the same time promotes thedissolution of the peptide subunit in aqueous media. However, uponprotonation of the carboxylate moieties, not only does the unfavorableintermolecular electrostatic interactions vanish, but a multitude ofattractive side chain-side chain hydrogen bonding interactions becomeoperative. Furthermore, at acidic pH the peptide subunit displays lowersolubility in aqueous media. This further contributes to an orderedphase transition toward self-assembled particles of cyclic peptidetubes. It is therefore concluded that since inter-subunit hydrogenbonding interactions provide the major driving force in the assemblyprocess, and considering that only in the stacked configuration can thepeptide subunits enjoy maximum possible number of hydrogen bondingcontacts, protonation of the carboxylate moieties strongly biases thesystem toward self-assembly.

Controlled acidification of alkaline peptide solutions is disclosed totrigger spontaneous self-assembly of peptide subunits into rod-shapedcrystals, as illustrated in FIGS. 18A and 18B. Transmission electronmicroscopy indicates that each particle is an orcanized bundle ofhundreds of tightly packed cyclic peptide tubes. Low dose cryomicroscopy of the particles reveals longitudinal striations with spacingof 14.9 Å along the long axis of the crystal, as expected, for thecenter to center spacing for closely packed cyclic peptide tubes, asillustrated in FIGS. 18C and D. As illustrated in FIG. 18E, electrondiffraction patterns display 4.73 Å axial periodicity demonstrating thateach cyclic peptide tube is made up of stacked rings with intersubunitdistances corresponding to an ideal anti-parallel β-sheet structure asillustrated in FIG. 9B, e.g., Salemme, F. R. (1983) Prog. Biophys.Molec. Biol. 42, 95-133 and Stickle, D. F. et al. (1992), J. Mol. Biol.226, 1143-1159. Given the thin rod shape of the crystals—on average 10to 30 micron in length by 100 to 500 nanometer in width, it isreasonable to assume that crystals would lay on the supporting EM carbonfilm along the long axis and either of the eab or ac faces. However, allmicrographs and diffraction patterns show similar diffractionintensities and nearly identical lattices. This suggests that the a andb axes are identical in size within experimental error. Furthermore,given the highly symmetric nature of the octapeptide, it seemsreasonable that the similarity of the views reflects the symmetry of themolecule. Electron diffraction patterns showed a unit cell having ameridional spacing of 14.92±0.08 Å and an axial spacing of 4.73±0.02 Åwith an angle of 99.2±0.5°. The diffraction patterns did not show anysymmetry other than the center of symmetry due to Friedel's law. Thepatterns extend to 1.5 Å and occasionally to 1.2 Å indicating that thecrystals are highly ordered. Considering the above observations, itfollows that the lattice is trigonal with a=4.73 Å, b=c=15.1 Å, α=90°,and β=γ=99°.

Although many conformers are possible using reasonable peptide backboneφ, ψ angles, only one fits the box defined by the unit cell-thedisk-shaped flat conformation where all side chains extend out in theplane of the disk. Other conformations are puckered and the irregularpresentation of side chains and backbone conformation prevent thepacking of peptide subunits to within 4.7 Å due to prohibitive van derWaals contacts. The only way to pack two peptides to within 4.7 Å is tohave the backbone carbonyl functionalities hydrogen bondintermolecularly to the nitrogen amide moieties of the opposite chainand position the side chains in the plane of the peptide backbone inorder to avoid unfavorable side chain-side chain steric interactions.Furthermore, due to the use of alternating D- and L-amino acid residues,the most favorable intermolecular hydrogen bonds form when the disks arestacked on top of one another in an anti-parallel fashion giving rise tolocal two fold symmetry, as illustrated in FIG. 20. The model built inthis way, using program XtalView disclosed by McRee, D. E. (1992) J.Mol. Graphics 10, 44-46, has unit cells which might at first glance seemtwice as large—9.5×15.1×15.1—which would require a spacing of 9.4 Å inthe diffraction pattern. However, when diffraction patterns arecalculated every other level of h cancels out due to the pseudo-symmetryof the dimer. That is, the ring flipped over is nearly equivalent to theunflipped ring. Several other packing were tried but none weresuccessful in forming a three-dimensional lattice without spacialoverlaps and simultaneously fit the spacing indicated by the electrondiffraction patterns. Cells where the local two-fold of the dimer iscrystallographic were also considered but then the lattice could eithernot be modeled or the diffraction pattern had to have a mirror planewhich of course is not observed. One other possibility is to considerthat the observed diffraction pattern shows a diagonal of the latticeinstead of a principle axis. This can be easily dismissed since itrequires a substantially smaller unit cell. It should be pointed outthat the volume of the unit cell in our proposed model is just largeenough to contain the octapeptide and anything smaller can notphysically hold the entire mass of the octapeptide. Therefore, theobserved electron diffraction pattern must be due to the principle axis.Finally the crystalline model was checked for bad contacts using XPLOR,a program disclosed by Brunger, A. T. et al. (1987) Science 235, 485(molecular dynamics calculations on the three-dimensional crystallinelattice). No bad contacts were found and the overall energy of the modelwas low. Most importantly, when the three-dimensional model was used tocalculate structure factors, the patterns produced compared veryfavorably with the electron diffraction patterns thus stronglysupporting the efficacy of the proposed model.

Involvement of intermolecular hydrogen bonding networks in tube assemblyis also evident by FT-IR spectroscopic analysis¹⁹, FIGS. 19A-C. Inalkaline solutions, the monomeric peptide subunit displays an amide-Iband at 1632 cm⁻¹ consistent with the cyclic structure of the backbone.Moreover, the bands at 1568 and 1670 cm⁻¹ are typical solvent exposedcarbonyl stretching frequencies for glutamate and glutamine side chainfunctionalities, respectively. However, upon self-assembly, cyclicpeptide tubes display characteristic features of a hydrogen bondedb-sheet structure. Not only is the appearance of amide I bands at 1628and 1688 cm⁻¹ and the amide II band at 1540 cm cm⁻¹ consistent with theexpected backbone structure, but the observed NH stretching frequency at3277 cm⁻¹ also strongly supports formation of a tight network ofbackbone-backbone hydrogen bonding. Given the tight backbone-backboneintersubunit interactions, as determined by the above analysis of theelectron diffraction patterns, it is possible to use Krimm's correlationto estimate intermolecular hydrogen bonding distance from the observedfrequency of the N—H stretch, e.g., Krimm, S. et al., in Advances inProtein Chemistry (eds Anfinsen, C. B., Edsall, J. T. & Richards, F. M.)181-364 (Academic Press, Orlando, 1986). The observed frequency of NHstretching mode approximately correlates to an average intersubunit N—Odistance of 2.85 Å or an intersubunit distance of 4.71 Å. This value isin close agreement with the value of 4.73 Å obtained from electrondiffraction patterns. It is noteworthy to point out that the IR spectrumof the cyclic peptide tubes also closely resemble the ones reported forGramicidin A—a naturally occurring linear peptide composed ofhydrophobic amino acids with alternating D- and L-configuration which isknown to form dimeric b-helical transmembrane ion channel structures,e.g., Wallace, B. A. et al. (1988), Science 241, 182-187 and Langs, D.A. (1988), Science 241, 188-191. Gramicidin A has amide I bands at 1630,1685 cm⁻¹, an amide II band at 1539 cm⁻¹, and an NH stretching frequencyat 3285 cm⁻¹, consistant with Naik, V. M. et al. (1986), Biophys. J. 49,1147-1154.

In summary, an example of a new class of synthetic tubular materials isdisclosed. The general strategy described should allow for the designand synthesis of a wide range of tubular structures with specifiedinternal diameters and surface characteristics. The availability of sucha simple strategy for the design of open ended tubular structures shouldundoubtedly lend itself to a wide range of applications, e.g.,Whitesides, G. M. et al. (1991), Science 254, 1312-1319 and ozin, G. A.(1992) Adv. Mater. 4, 612-649. Such tubular materials could be designedto mimic biological channels and pore structures, used to study physicaland chemical properties of confined molecules, control growth andproperties of inorganic and metallic clusters, or design novel opticaland electronic devices.

Use of a Nanotube as a Carrier:

The cyclic peptide tubes disclosed in Example 1 may be assembled in thepresence of hydrogen peroxide. After tube assembly and particleformation, the mixture is centrifuged in order to pellet the particles.The pelleted particles are washed by a further centrifugation step andthen combined with the reagents of bioluminescent assay designed to testfor the presence of hydrogen peroxide. Bioluminescence is seen to beconfined to the pelleted fraction. This demonstrates that the cyclicpeptide tubes can encapsulate hydrogen peroxide within their channelregion but that the hydrogen peroxide slowly leaks by diffusion fromsuch channel region into the external media.

Self-Assembled Nanotube with a 13 Å Pore:

The pore size of self-assembled organic nanotubes can be simply adjustedby the ring size of the peptide subunit employed. The internal diameterof the nanotube ensembles can be rigorously controlled simply byadjusting the ring size of the peptide subunit employed. Atwelve-residue cyclic peptide structure, i.e., the thirty six-memberedcyclic peptide subunit cyclo[-(Gln-D-Ala-Glu-D-Ala)₃-] (Sequence No.: 8)has been designed and shown to undergo a proton-induced self-assemblyprocess to produce highly ordered nanotubular objects having a uniform13 Å internal van der Waals diameter. These nanotubes have beencharacterized by IR spectroscopy, low-dose electron microscopy, and theanalysis of electron diffraction patterns. The ability to designspecifically sized tubular nanostructures is expected to have importantapplications in catalysis, inclusion chemistry, and molecularelectronics. Formation of the tubular structures is supported by highresolution imaging using cryo electron microscopy, electron diffraction,Fourier-transform infrared spectroscopy, and molecular modeling.

According to the disclosed design principles, cyclic peptide structureswhich are made up of an even number of alternating D- and L-amino acidresidues can adopt or sample a flat ring-shaped conformation in whichall backbone amide functionalities lie approximately perpendicular tothe plane of the ring structure. In this conformation, the peptidesubunits can stack, under favorable conditions, to furnish a contiguoushydrogen bonded hollow tubular structure (FIG. 22). The internaldiameter of the nanotube ensemble can, in principle, be tailored byadjusting the ring size of the peptide subunit employed. In this studywe will describe the largest pore diameter peptide-based nanotubestructure thus far constructed by utilizing the following thirtysix-membered ring peptide subunit cyclo[-(Gln-D-Ala-Glu-D-Ala)₃-]. Thedesign principles and the self-assembly strategy employed in this studyis similar to the one described previously¹. The requisite peptidesubunit was synthesized on a solid-support, according to the method ofP. Rovero et al. (Tetrahedron Lett., (1991), vol. 32, pages 2639-2642)and characterized by mass spectrometry and ¹H NMR spectroscopy.Controlled acidification of alkaline solutions of the peptide subunitupon standing afforded rod shaped crystalline materials, as indicatedabove. Transmission electron microscopy indicates that each particle isan organized bundle of tightly packed nanotubes (FIGS. 23A-B). Low dosecryo microscopy, according to the method of M. Adrian et al. (Nature(1984), vol. 308, pages 32-36) and of R. A. Milligan et al.(Ultramicroscopy (1984), vol. 13, pages 1-10) revealed longitudinalstriations with spacing of approximately 25 Å as expected for the centerto center spacing for closely packed nanotubes (FIGS. 23A-B). Electrondiffraction patterns display axial spacing of 4.80 Å which is inagreement with the peptide stacking and the formation of tight networkof hydrogen bonded b-sheet type structure. The Meridonial spacing in theelectron diffraction patterns display spacing of 12.67±0.06 Å and21.94±0.05 Å characteristic of a hexagonal body centered packing ofnanotubes. Hexagonal lattice resulting from the close packing ofcylinders of radius r displays the characteristic two principle latticeplanes of radius r and r such as the one observed here (r=12.67 Å andr=21.94 Å). The periodicity in this packing produces diffraction spotsat 1/r, 2/r, and so on, and at 1/r, and 2/r, and so on. The observedelectron diffraction patterns on the meridional axes extend to thirdorder reflections (4.1 Å) signifying the ordered and crystalline stateof the nanotube particles. The diffraction patterns also showed a unitcell with an angle of 99° and no other symmetry than the center ofsymmetry due to Friedel's law.

A three-dimensional model of the nanotube structure was built using theparameters obtained from the electron diffraction patterns-unit cellwith a=9.6 Å (2×4.80 Å for the antiparallel dimer), b=c=25.66 Å(2×12.67÷ cos 9), α=120°, and β=γ=99°. The model shows structure factorssimilar to the patterns observed in the electron diffraction thussupporting the proposed three-dimensional model. Involvement ofintermolecular hydrogen bonding network in the tube assembly is alsosupported by FT-IR spectroscopic analysis according to the method of S.Krimm et al. (Advances in Protein Chemistry; Anfinsen, C. B., Edsall, J.T.; Richards, F. M. Eds.; Academic Press: Orlando, 1986, pages 181-364).Nanotubes display characteristic IR features of a β-sheet structuresignified not only by the amide I bands at 1626 cm⁻¹ and 1674 cm⁻¹ andan amide II band at 1526 cm⁻¹, but also by the observed NH stretchingfrequency at 3291 cm⁻¹ supporting formation of a tight network ofhydrogen bonds. The IR spectrum is very similar to other nanotubes andclosely resembles that of crystalline Gramicidin A which is known toform dimeric β-helical structures. Gramicidin A has amide I bands at1630, 1685 cm⁻¹, an amide II band at 1539 cm⁻¹, and an NH stretchingfrequency at 3285 cm⁻¹. (V. M. Naik et al. in Biophys. J. (1986), vol.49, pages 1147-1154.) The observed frequency of NH stretching modecorrelates to an average intersubunit distance of 4.76 Å which is inclose agreement with the value of 4.80 Å obtained independently from theelectron diffraction patterns.

Artificial Transmembrane Ion Channels from Self-Assembling PeptideNanotubes:

Artificial membrane ion channels may be constructed using self-assembledcylindrical β-sheet peptide architecture. The construct describeddisplays an efficient channel-mediated ion transport activity with ratesexceeding 10⁷ ions.sec⁻¹ rivaling that of many naturally occurringcounterparts. Such molecular assemblies are expected to have potentialutility in the design of novel cytotoxic agents, membrane transportvehicles, and drug delivery systems.

According to the design principles herein disclosed, cyclic peptidestructures made up of an even number of alternating D- and L-amino acidresidues can adopt a flat ring conformation and stack, under favorableconditions, to furnish a contiguous hydrogen bonded hollow tubularstructure. Therefore, an ensemble made up of eight to ten subunits, eachseparated by the expected intersubunit distance of 4.7 to 5.0 Å anddecorated with appropriate hydrophobic surface residues, would be longenough to span the thickness of average biological lipid membranes (FIG.2). The eight residue cyclic peptide cyclo[-(Trp-D-Leu)₃-Gln-D-Leu-](Sequence No.: 9) was designed for the purpose in hand. It is composedof alternating L-tryptophan and D-leucine side chain moieties with theexception of one L-glutamine residue introduced mainly to simplify thepeptide synthesis. It is demonstrated that the channel structures havingan aqueous pore of approximately 7.5 Å in diameter would formspontaneously upon incorporation a sufficient concentration of thepeptide monomer in lipid bilayers. The driving force for theself-assembly of the channel structure is primarily provided by theenthalpic contribution of a large number of hydrogen bondinginteractions which are favored in the low dielectric constant medium oflipid bilayers and by the increase in the lipid chain entropy arisingfrom side chain-lipid interactions. (See D. M Engelman et al. in Cell(1981), vol. 23, pages 411-422; L. C. Allen in Proc. Natl. Acad. Sci.USA (1975), vol. 72, pages 4701-4705; and D. M. Engelman in Annu. Rev.Biophys. Chem. (1986), vol. 15, pages 321-353). In short, it isdemonstrated that the designed flat ring-shaped cyclic peptide is notonly structurally predisposed toward intermolecular interaction, but isalso energetically favored to self-assemble, in the lipid bilayerenvironment to furnish the desired transmembrane channel structure. Thefollowing studies using a variety of spectroscopic techniques, lipidvesicle model systems, and single ion channel recordings support thevalidity of the above design hypothesis.

Addition of the peptide subunit to aqueous liposomal suspensions effectsa rapid partitioning of the subunit into the lipid bilayers and itsspontaneous self-assembly into ion transport-competent membrane channelstructures. Incorporation of the peptide subunit into lipid bilayersusing large unilamellar vesicles has been established by absorption andfluorescence spectroscopy. Formation of the hydrogen-bondedtransmembrane channel structure in phosphatidylcholin liposomes has beensupported by FT-IR spectroscopy (FIG. 24). The observed amide-I band at1624 cm⁻¹ is not only similar to the carbonyl stretching frequenciesfound in other nanotube structures disclosed herein, but is alsoconsistent with the infrared spectrum of gramicidin A in similar lipidbilayers. (E. Nabedryk et al in Biophys. J. (1982), vol. 38, pages243-249. Furthermore, the observed N—H stretching frequency at 3272 cm⁻¹strongly supports the formation of a tight network of hydrogen bondswith an average intersubunit distance of 4.7 Å.

Formation of transmembrane channels was also inferred from its highlyefficient proton transport activity. Vesicles were prepared having pH6.5 inside and pH 5.5 in the outside bulk solution. The collapse of theimposed pH gradient in these vesicles, upon formation of the putativetransmembrane channel structure, was studied by monitoring thefluorescence intensity of an entrapped pH-sensitive dye. (V. E.Carmichael et al, in J. Am. Chem. Soc. (1989), vol. 111, pages 767-769).As shown in FIG. 25, addition of the peptide to such vesiclessuspensions causes a rapid collapse of the pH gradient. Unilamellarvesicles were prepared by the reverse-phase evaporation using DPPC,OPPC, cholestrol in the ratio of 1:1:2 in a solution containing5(6)-carboxyfluorescein (20 mm in phosphate/saline buffer: 137 mM NaCl,2.6 mM KCl, 6.4 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH 6.5) according to themethod of F. Szoka et al. in Proc. Natl. Acad. Sci. USA (1978), vol. 75,pages 4194-4198. Liposomes were then sized by multiple extrusionsthrough Nucleopore® polycarbonate membranes (10 times, 50 psi, using 0.8and 2×0.4 micron filter stacks) and the untrapped5(6)-carboxyfluorescein was removed by size exclusion chromatography(Sephadex G-25 column 1×30 cm) using the same phosphate/saline bufferaccording to the method of F. Olson et al. in Biochim. Biophys. Acta(1979), vol. 557, pages 9-23. Vesicles formed in this way areapproximately 150 nanometer in diameter as determined by electronmicroscopy. (R.R.C. New, Ed. Liposomes, Oxford university Press, 1990).In each experiment, 70 ml of the stock vesicle solution (3.5×10⁻³ M inphospholipids) was added to pH 5.5 buffer (1.3 ml, 137 mM NaCl, 2.6 mMKCl, 6.4 Na₂HPO₄, 1.4 KH₂PO₄) and placed in a 1 cm quartz cuvett insidea stirring thermojacketed sample holder of the florescence instrumentand equilibrated at 25° C. for 15 minutes with gentle stirring. To thecuvett, through an injector port, 25 ml of the channel forming compoundsin DMSO was added with continuous fluorescence monitoring at 520 nm(excitation at 470 nm). The observed data were then normalized forcomparison into the fractional change in fluorescence((I₀-I_(t))/(I₀-I_(∞))) (V. E. Carmichael et al, in J.Am. Chem. Soc.(1989), vol. 111, pages 767-769)). According to these experiments, theapparent ion transport activity of cyclo[-(Trp-D-Leu)₃-Gln-D-Leu-] issimilar to, if not higher than, that of gramicidin A and amphotericin B(FIG. 25). The lipid bilayers used were formed on the tip of patchpipettes using a mixture of synthetic POPE: POPS (4:1)(1-palmitoyl-2-oleoyl-Sn-glycero-3-phosphatidylethano lamine and-serine). Five to 10 ml of the peptide solution (1.0×10⁻⁷ or 2.0×10⁻⁶ Min 25% DMSO in buffer solution containing 500 mM NaCl or KCl, 5 mMCaCl₂, 10 mM HEPES, pH 7.5) was added to 150 ml subphase volumeresulting in spontaneous partitioning of the peptide into the membrane.Ion channels formed spontaneously after peptide was added to thesubphase of lipid bilayers. Ion channel activity was observed in 14 outof 22 membranes under symmetrical solutions of 500 mM NaCl or KCl, 5 mMCaCl₂, 10 mM HEPES, pH 7.5. Data acquisition and analysis were performedon a Gateway 2000/486 computer using pClamp software package and TL-1labmaster interface. Acquisition rate was 0.1 ms and data were filteredat 2 kHz.

Control studies, monitoring the release of 5(6) carboxyfluorescein dye,indicated that the collapse of the pH gradient was not due to therupturing of the liposomes nor due to the small amounts of organicsolvents (<2% DMSO) employed in these studies. Furthermore, the controlpeptide cyclo[-(Gln-D-Leu)₄] which lacks the appropriate surfacecharacteristics for partitioning into the lipid bilayers, but otherwisequite similar in design to the channel forming peptide described above,does not display any ion transport activity under similar conditions.The second control peptide cyclo[-(^(Me)N-D-Ala-Phe)₄-] which has thedesirable hydrophobic surface characteristics but lacks the propensityfor participating in extended hydrogen bonding network, was alsodesigned and tested for ion transport activity. The peptide designincorporates a novel N-methylation strategy on one face of the ringstructure which predisposes the subunit toward a dimeric cylindricalstructure (Ghadiri, M. R., Kobayashi, K., Granja, J. R., Chadha, R., andMcRee, D. E. manuscript in preparation). Such a dimeric cylindricalensemble is approximately 10 Å thick and can not span the lipid bilayer.Although the peptide has been shown to partition effectively into lipidbilayers, it does not promote proton transport activity in the abovevesicle experiments. Together, these experiments suggest that not onlythe hydrophobic surface characteristic of the channel forming peptide isan important factor, but also the peptide subunit must be able toparticipate in extended hydrogen-bonded stacking interactions to producechannel structures long enough to span the lipid bilayer.

The designed transmembrane ensemble also shares importantcharacteristics with natural ion channel formers such as gramicidin Aand amphotericin B. First, the peptide shows concentration dependenceeffects on the rate of channel formation (data not shown). Second, whenlow concentrations of the channel forming peptide is used in the aboveproton transport experiments, only part of the vesicle population goesto equilibrium very fast. This phenomenon reflects the statisticaldistribution of the channel forming species among the vesiclepopulation—only part of the population has enough channel-formingmolecules to form permeable competent structures in an all-or-none typeof a process. Unlike “ion carriers” such as monensin and valinomycinwhich bind to metal ions and partition between aqueous-phase and thelipid-phase in order to establish ion equilibrium across the membrane,channel forming species at low concentrations (the designed peptidehere, amphotericin B, gramicidin A, and others) because of theirinability to defuse back out of the membrane, can not penetrate othervesicles and unlike ionophores can not easily establish proton or ionequilibrium in all vesicles present in solution. Therefore, the observedrapid proton efflux in the above types of experiments simply reflectsthe rate limiting step of peptide diffusion into the lipid bilayer andself-assembly into ion-transport competent channel structures and doesnot reflect the actual rate of channel-mediated ion transport which canoccur on a much faster time scale (Vide infra).

The ultimate test in establishing and quantitating the transportefficiency of a membrane channel structure is to measure its singlechannel conductance using micro patch clamp techniques. (B. A.Suarez-Isla et al., Biochemistry (1983), vol. 22, pages 2319-2323.)Observing a high throughput rate of ions demonstrates ion channelformation and is a diagnostic feature distinguishing ionic channelmechanisms from those of other ion transport devices such as ioncarriers. (P. Leuger, Angew. Chem. (1985), vol 97, page 939); and Angew.Chem. Int. Ed. Engl. (1985), vol. 24, pages 905-923.) Single channelconductances, using planar lipid bilayers with peptide concentrations inthe range of 10⁻⁷ M in the subphase, are approximately 55 pS (picoSiemens) in 500 mM NaCl and 65 pS in 500 mM KCl (FIG. 26). Higherconductance found in KCl as compared to NaCl is consistent with theexpected weak ion selectivity of the 7.5 Å pore structures and reflectsthe relative mobility of Na⁺ vs. K⁺ ions in solution. Therefore, a 55 pSchannel in NaCl and a 65 pS channel in KCl are likely to arise from thesame structural entity (same number of stacked rings). In addition,single channel conductance was shown to be independent of the appliedvoltage in the measured range of 10-100 mV. The actual rate ofchannel-mediated ion transport is therefore a prodigious 2.2×10⁷ion.sec⁻¹ for K⁺ and 1.8×10⁷ ion.sec⁻¹ for Na⁺ which is almost threetimes faster than that of gramicidin A under similar conditions. (E.Bamberg et al. in Biochim. Biophys. Acta (1974), vol. 367, pages127-133.

The strategy described here also allows for the design of transmembranechannel structures with larger pore diameters for use in “molecular”transport across lipid bilayers and as such it should provide apotential vehicle for drug delivery into living cells and may find usein antisense and gene therapy applications.

Channel-Mediated Transport of Glucose Across Lipid Bilayers:

The design, synthesis, and characterization of an artificialtransmembrane pore structure capable of mediating transport of glucoseaccross lipid bilayers is disclosed. The design strategy is based on thepropensity of a novel 10-residue cyclic peptide subunitcyclo[-Gln-(D-Leu-Trp)₄-D-Leu-] (Sequence No. 10) toward spontaneousself-assembly in lipid bilayers to form 10 Å van der Waals aqueous porestructures. Molecular modeling indicated that for the passage of glucosethrough the cylindrical cavity of the tubular transmembrane structure,the internal van der Waals pore diameter of greater than 9 Å isrequired. Therefore for the task in hand, a ten-residue peptide subunitis employed which upon self-assembly can produce tubular ensembleshaving a uniform 10 Å internal diameter (FIGS. 27A-B).

The ten-residue peptide subunit employed in the present study,cyclo[-Gln-(D-Leu-Trp)₄-D-Leu-], is made up of largely tryptophan andleucine residues to favor its partitioning into and self-assembly inlipid bilayers. It was synthesized on solid support⁵ and characterizedby ¹H NMR spectroscopy and mass spectrometry. Addition of the peptidesubunit to aqueous suspensions of large unilamellar liposomes effects arapid incorporation of the peptide into the lipid bilayer. This has beensupported by absorption and fluorescence spectrophotometry and gelpermeation studies. Formation of a hydrogen bonded transmembrane channelstructure in phosphatidylcholin liposomes has been established by FT-IRspectroscopy⁶. The observed amide-I band at 1,625 cm⁻¹ and the N—Hstretching band at 3,272 cm⁻¹ are similar to those of the previouslycharacterized peptide nanotubes^(2a,4) and strongly support theformation of a tight network of b-sheet-like hydrogen bonded structureswith an average intersubunit distance of 4.8 Å. Formation oftransmembrane channels was also inferred from its remarkably high iontransport efficiencies (>10⁷ ions.sec⁻¹) as indicated by single ionchannel recordings using micro patch clamp techniques.

Glucose transport activity was studied in isotonic solutions usingglucose entrapped unilamellar lipid vesicles. Unilamellar vesicles, ˜150nm in diameter, were prepared by the reverse-phase evaporation methodusing 1,2-dipalmitoyl-Sn-glycero-3-phosphatidylcholine (DPPC),1-palmitoyl-2-oleoyl-Sn-glycero-3-phosphatidylcholine (POPC),1-palmitoyl-2-oleoyl-Sn-glycero-3-phosphatidylserine (POPS), andcholesterol in the ratio 1:1:0.1:1 in a solution containing 50, 100,150, or 200 mM D-glucose, 100 mM NaCl, and 50 mM Tris buffer pH 7.5,according to the method of F. Szoka et al. in Proc. Natl. Acad. Sci. USA(1978), vol. 75, pages 4194-4198. Liposomes were gel-filtered usingSephadex G-25 in an isotonic buffer containing 50, 100, 150, or 200 mMsucrose, 100 mM Nacl, and 50 mM Tris buffer pH 7.5. The liposomepreparation was stored at 4° C. and used within 24 hours of thesynthesis. The transport phenomenon was monitored spectrophotometricallyat 340 nm for the production of NADPH using an enzyme coupled assay(FIG. 28), according to the method of S. C. Kinsky in Methods inEnzymology; Fleischer, S., Packer, L., Eds; Academic Press: London,1974; vol. 32, pp 501-513. All experiments were performed on aSpectronic-3000 photodiode-array spectrophotometer using 3 ml quartzcuvettes placed in the thermojacketed multiple cell holder and held at27° C. In a typical experiment the following solutions were sequentiallyplaced in the cuvettes: 750 ml of buffer (300 mM NaCl, 50 mM Tris pH7.5, 3.5 mM MgCl₂, and 0.15 mM CaCl₂), 500 ml of the enzyme solution (8units.ml⁻¹ of hexokinase, 16 units.ml⁻¹ of glucose-6-phosphatedehydrogenase, 2.5 mM ATP, 1.3 mM NADP all dissolved in 200 mM NaCl, 50mM Tris pH 7.5, 3.5 mM MgCl₂, and 0.15 mM CaCl₂), and 75 ml of the stockliposome solution⁸ (2.6×10⁻³ M in phospholipids). Total glucose contentin each cuvette was determined by Triton X-100 treatment. Transport wasinitiated by the addition of an appropriate amount (5, 7.5, 10, 15, or20 ml) of the peptide solution (1 mM in DMF) to the reference and thesample cuvettes (hexokinase was omitted from the reference sample). Formeasuring the background (nonspecific glucose leakage from theliposomes) the sample was prepared in an identical fashion exceptappropriate amounts of DMF were added in place of the channel formingpeptide. The production of NADPH was monitored at 340 nm for 1.5 h at 90second time intervals. Because of the high catalytic efficiencies of theenzymes employed, the rate of NADPH production is directly proportionalto the rate at which glucose is released from the liposomes. Thetransport of glucose initiated by the addition of various amounts of thechannel forming peptide to the glucose-entrapped liposomes follows afirst order rate profile with an apparent rate constants of 1.2±0.09,0.74±0.1, 0.48±0.05, and 0.18±0.02 mol(glucose)/mol(peptide)/min⁻¹ forliposomes having initial glucose concentrations of 200, 150, 100, and 50mM, respectively (FIG. 29). The apparent rate of glucose transport is,in all likelihood, a gross underestimation of the actual rate of channelmediated transport because only a minute fraction of the total number ofpeptides incorporated in the lipid bilayer are assembled, at a giventime, in the form of active transmembrane channel structures. Unlikecarrier-mediated transport which must display Michael is-Mentensaturation kinetics, the observed linear relation between transport rateand glucose concentration strongly supports a simple transmembranechannel-mediated diffusion process. (W. D. Stein in Channels, Carriers,and Pumps, Academic Press: San Diego, 1990.) Control studies, monitoringthe release of entrapped 5(6)-carboxyfluorescein under similarconditions, established that the transport of glucose was due to neitherthe rupturing of the liposomes nor the small amounts of DMF (<2%)employed in these studies. (See: N Jayasuriya et al., in J. Am. Chem.Soc. (1990), vol. 112, pages 5844-5850; and J. N. Weinstein et al., inScience (1977), vol. 195, pages 489-492.) Furthermore, neithergramicidin A, a well-known naturally occurring ion channel formingpeptide with an internal diameter of approximately 4.5 Å, nor the verysimilar ion channel forming peptidecyclo[-Gln-(D-Leu-Trp)₃-D-Leu-],disclosed herein to form channels withapproximately 7.5 Å internal diameter, display any glucose transportactivity under similar assay conditions. Together, this demonstratessize-selective pore-mediated transport of glucose.

Carped Nanotubes:

Another such feature has to do with channel capping—the process by whichthe self-assembled molecular tube is terminated. It is evident from FIG.4 that the subunits at the channel openings, i.e., at the “cap”positions, are unique with respect to their mode of interaction with theother subunits as well as the micro-environment in which they reside.The peptide subunits at the cap position participate inbackbone-backbone hydrogen bonding with only one other subunit and ononly one side of the backbone structure. The cap subunits also reside inthe amphiphilic micro-environment of the lipid-water interface.

The key structural requirement for producing a multiple ring-stackedtubular structure is the spatial disposition of the backbone hydrogenbond donor and acceptor sites on both faces of the peptide ringstructure. However, if the cyclic peptide subunit is devoid of hydrogenbond donation from one face of the ring structure through the blocking,e.g., alkylation of backbone amide nitrogen functionalities of one ofthe chiral moieties present, such a cyclic peptide subunit cannotparticipate in an extended hydrogen bonding network, but serves to cap atubular structure.

Such selectively alkylated cyclic peptides in non-polar solution arepredisposed to dimerization. However, the addition of such selectivelyalkylated cyclic peptide subunits to the aforementioned ring-stackedtubular structure in an appropriate solvent permits capping ortermination of the ring-stacked tubular structures by the monomeric,selectively alkylated cyclic peptides.

One such preferred cyclic peptide is the eight-residue cyclic peptidecyclo[-(L-Phe-D-^(Me)NAla)₄-] shown in FIG. 15. The most meaningful wayof classifying the various amino acids is on the basis of the polarityof their R groups in water near pH 7. There are four main classes: (1)nonpolar or hydrophobic, (2) polar but uncharged, (3) positivelycharged, (4) negatively charged. The nonpolar or hydrophobic groupincludes amino acids with aliphatic R-groups, such as alanine, leucine,isoleucine, valine, and proline, amino acids with aromatic ringR-groups, such as phenylalanine and tryptophan, and one amino acid witha sulfur-containing R-group, methionine.

Molecular modeling indicated that methylation of backbone amide nitrogenfunctionalities at all alanine residues would be sufficient toeffectively prevent one face of the putative peptide ring structure fromparticipating in intermolecular hydrogen bonding and ring stackinginteractions. The peptide was also designed to have an allowed symmetryfor favorable intermolecular packing interactions in the solid-statethus permitting its detailed structural characterization by X-raycrystallographic techniques Amino acids having hydrophobic R-groups werechosen for this Example so as to make the subunit and the resultingdimer ensemble soluble in nonpolar organic solvents. However, the choiceof the R group to any given instance depends on the desired solubilitycharacteristics of the end product.

The linear form of the target sequence H₂N-(L-Phe-D-^(Me)NAla)₄—CO₂H wassynthesized according to standard solid-phase methods describedhereinabove and then cyclized in solution to furnish the desired cyclicpeptide subunit using the following procedure. A solution of the linearpeptide in DMF (1 mM) was treated with TBTU(2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate,3 mM), HOBt (1-Hydroxybenzotriazole, 3 mM) and DIEA(diisopropylethylamine, 1% v/v) at 5° C. for 12 h to give the desiredcyclic peptide monomer, after reverse-phase HPLC purification, in 70%yield, which can then be used for capping or terminating peptidenanotubes.

The ¹HNMR spectrum of the produced peptide subunit in polar solvents,such as deuterated methanol or dimethyl sulfoxide (DMSO), displaysmultiple slow-exchanging conformational isomers due to the well-knownpropensity of secondary amides toward cis-trans isomerization. Variabletemperature NMR experiments in DMSO indicate cis-trans conformationalactivation barriers on the order of 16 to 17 kcal.mol⁻¹. However, innonpolar solvents such as carbon tetrachloride (CCl₄) ordeuterochloroform (CDCl₃) the peptide exists in an all transflat-ring-shaped backbone conformation which is in dynamic equilibriumwith the expected dimeric cylindrical ensemble. The monomeric peptidesubunit displays a temperature independent (from −40 to 55° C. in CDCl₃)and highly symmetrical ¹H NMR spectrum excluding the possibility of anintramolecularly hydrogen bonded conformation The preponderance of aflat ring-shaped backbone conformation is also indicated by the observed7.5 Hz J_(NH—CaH) coupling constant. Intermolecular hydrogen bondinginteractions producing the stacked dimeric ensemble are signified by theexpected downfield shift of the phenylalanine N—H backbone resonancefrom 6.98 to 8.73 ppm (J_(NH—CaH)=8.5 Hz) and are unequivocallyestablished by the observed exchange and NOE cross peaks in its ROESYspectrum.

ROESY experiments were performed on a Bruker AMX-500 with 300 ms spinlock (mixing) time using Bruker's standard pulse program. Data wereprocessed using FELIX software. Time domain data was apodized usingskewed sine-bell squared window functions. Zero-filling was used toobtain the final data size of 1024×1024 complex matrix. A. Bax, D. G.Davis, J. Magnetic Resonance 1985, 63, 207-213.

Formation of a tight hydrogen bonded ensemble with an averageintersubunit N—O distance of 2.95 Å is also evidenced by the appearanceof an N—H stretching band in the infrared spectrum at 3309 cm⁻¹. Asexpected, the self-assembly process displays concentration and solventdependent spectra with the association constants K_(a)(CCl₄)=1.4×10⁴M⁻¹and K_(a)(CDCl₃)=1.26±0.13×10³M⁻¹ at 293 K.

The association constants reported are the lower limits due to thepresence of small amounts of included water in the peptide samples. Whenwater is rigorously excluded (4 Å molecular sieves) the associationconstant K_(a)(CDCl₃)=1260 M⁻¹ is approximately doubled toK_(a)(CDCl₃)=2540 M⁻¹. The K_(a)(CCl₄) reported was performed in amixture of 84% CCl₄ and 16% CDCl₃ for solubility reasons.

Variable temperature studies (van't Hoff plots) establish the followingthermodynamic parameters for the dimerization process in chloroform:ΔCp=−203.1 cal.K⁻¹.mol⁻¹, ΔH⁰ ₂₉₈=−11.0 kcal.mol⁻¹, and ΔS⁰ ₂₉₈=−23.7e.u. which clearly supports the expected enthalpic contribution ofintermolecular hydrogen bonding interactions (0.5 to 0.7 kcal.mol⁻¹ foreach hydrogen bonding interaction) as the major driving force in theself-assembly process.

The above experiments indicate that the peptide subunit adopts a flatring-shaped solution conformation which is energetically favored towardring stacking and intermolecular hydrogen bonding interactions by 4.0 to5.6 kcal.mol⁻¹, depending on the solvent employed. It follows then thatan additive gain in free energy of stabilization is to be expected asthe number of ring-stacking interactions are increased. This isparticularly relevant to the self-assembled nanotubes and to thetransmembrane ion channel structures that can be produced.

Colorless prismatic crystals suitable for X-ray analysis were obtainedfrom the solutions of the peptide in water-saturated dichloromethane byvapor-phase equilibration with hexane. The crystal structure was solvedin the space group I422 with a final R-factor of 8.87%. Data werecollected on a Rigaku AFC6R diffractometer equipped with a copperrotating anode (Cu_(Ka)) and a highly oriented graphite monochromator.The structure was solved in the space group I422 with a final R-factorof 8.87% and weighted R-factor of 10.35% and the residual electrondensity of 0.64 eÅ⁻³ for 983 unique reflections with F>4.0 σ(F). Theunit cell parameters are a=b=16.78, and c=21.97 Å.

The solid-state structure is a cylindrical dimeric ensemble, analogousto the solution structure deduced from the ¹H NMR and FT-IR analyses,corroborating very well the previously calculated nanotube structuresderived primarily from the analysis of electron diffraction patterns.The dimeric ensemble is a combination of a flat ring-shaped cyclicpeptides subunit with backbone amide groups perpendicular to the planeof the ring structure and a crystallographic four-fold rotation axisparallel to the c axis passing through the center of the peptide ring.Two peptide subunits are closely stacked in an antiparallel orientationand are related by a two fold rotation along either a or b axis. Theb-sheet-like cylindrical ensemble is stabilized by eight intersubunithydrogen bonding interactions with an intersubunit N—O distance of 2.90Å. It is noteworthy that the distance of 2.95 Å inferred from theobserved NH stretching band at 3312 cm⁻¹ in the FT-IR spectrum isremarkably consistent with the crystallographic measurements. Thecylindrical ensemble has an approximate 7.5 Å van der Waals internaldiameter and a 450 Å³ volume. The tubular cavity is filled withpartially disordered water molecules, establishing the hydrophilicinternal characteristics of the peptide nanotube structures. Theensemble packs in the crystal in a body centered fashion to produce acontinuously channeled superlattice structure along the c axis. Theinterior surface characteristics of the channels alternate approximatelyevery 11 Å between the hydrophobic domains, created by the aromaticphenyl moieties, and the hydrophilic interior of the peptide cylindricalensemble. Water molecules near the hydrophobic domains are considerablymore disordered, displaying only a weak residual electron density. Theobserved water electron density is the time average of water moleculesbinding at multiple overlapping sites suggesting a facile movement ofloosely held water molecules within the cavity. This observation whichcan be attributed to the lack of a discrete, strong binding site(s), isan important attribute of the produced peptide nanotube structures andis believed to contribute to the remarkable transport efficiencies ofthe formed transmembrane ion channels.

The foregoing discussion and the accompanying examples are presented asillustrative, and are not to be taken as limiting. Still othervariations within the spirit and scope of this invention are possibleand will readily present themselves to those skilled in the art.

Gated Nanotubes:

Cyclic peptide tubes can also be employed as ion-gated membrane channelstructures. By the appropriate choice of the amino acid side chainmoieties one can tune, at will, the surface characteristics of theself-assembled cyclic peptide tubes. For the purpose of constructingmembrane channel structures, cyclic peptides are designed to havehydrophobic side chain moieties in order to ensure their insertion andself-assembly within the nonpolar environment of lipid bilayermembranes.

The anatomy of the tubular membrane channel structure is schematicallyshown in FIG. 2. It consists of approximately eight stacks ofanti-parallel peptide subunits which enables the channel structure tospan the thickness of the average biological lipid membrane-according toour previous electron diffraction studies on the self-assembled organicnanotubes, an average intersubunit distance of 4.8 to 5.0 Å is expectedfor such an extensively hydrogen bonded antiparallel β-sheet ensemble.The channel structure can form spontaneously upon the dissolution of asufficient concentration of the peptide monomer in the lipid bilayer.The driving force for the self-assembly of the channel structure isprovided by a) the enthalpic contribution of a large number of highlyfavorable and oriented hydrogen bonding interactions—each hydrogen bondin the nonpolar environment of the membrane is estimated to worth about5-6 kcal.mol⁻¹ (for a channel composed of eight 8-mer cyclic peptides,the hydrogen bond network consists of 56 highly cooperativeintermolecular hydrogen bonds), and b) by the increase in the lipidbilayer entropy arising from the side chain-lipid interactions. Thesehighly favorable energetic contributions easily compensate for the lossof entropy associated with the peptide self-assembly andself-organization. Furthermore, considering that only hydrophobicresidues are utilized in the peptide design, the unfavorable backbonedissolvation energy does not play a significant role in the assemblyprocess especially since the hydrophilic interior of the channelstructure is expected to be filled with a large number of interactingwater molecules. In short, such de novo designed cyclic peptides are notonly structurally predisposed toward intermolecular interaction, but arealso energetically favored to self-assemble, in the lipid bilayerenvironment, into artificial membrane channels. Furthermore, if needed,simple methods are available for linking the subunits together throughside chain-side chain covalent bond formation in order to obtain apermanently fused molecular channel structure.

The self-assembled channels have two important and unique structuralfeatures which are pertinent hereto. One feature is that the channelpore size can be easily tailored by choosing an appropriate ring sizefor the cyclic peptide subunit (FIG. 3). This allows for the design ofshape-selective membrane pore structures. The second feature, whichdeserves a brief explanation, has to do with channel gating-the processby which molecular transport across the channel is turned on or off. Itis evident from FIG. 4 that the two subunits at the channel entrance,i.e., the “cap” position, are unique with respect to their mode ofinteraction with the other subunits as well as the micro-environment inwhich they reside. The peptide subunits at the cap position participatein backbone-backbone hydrogen bonding with only one other subunit and ononly one side of the backbone structure. The cap subunits also reside inthe amphiphilic micro-environment of the lipid-water interface. Theseunique characteristics can be exploited for the design of gated membranechannels in the following fashion. In order to ensure segregation of thecap subunits from the other channel forming subunits, one face of thebackbone structure can be blocked from participating in inter-subunithydrogen bonding interactions simply by alkylating the backbone amidenitrogen functionalities at the homochiral residues. Such N-alkylatedspecies not only lack hydrogen bonding donor capability on one face ofthe disk structure but also the severe steric interaction imposed by theN-alkyl substituents effectively prevents the participation of thepeptide subunit in bi-directional hydrogen bonding stackinginteractions. Therefore, such N-alkylated subunits can only reside atthe cap positions. In addition, side chains capable of interacting withpolar lipid head groups may also be introduced to ensure its properpositioning at the lipid surface. As illustrated in FIG. 5, Amidenitrogen alkylation in addition to its hydrogen bonding disruptivecapability, also serves another important role, i.e., it provides asimple strategy for designing gated membrane channels. In general, awide variety of bi- or multi-dentate small-molecule receptors may beintroduced at the channel entrance through N-alkylation, as illustratedin FIG. 6.

FIG. 7, illustrates that this simple anchoring motif can be employedwith a number of transition metal ion binding sites. Channels structuresassembled in this way, are blocked (in the “off” position) in thepresence of transition metal ions toward molecular transport due to thesteric hindrance imposed by the metal ion-ligand interactions at thechannel entrance, as illustrate in FIG. 8. Such a strategy can also beemployed in the design of highly selective and sensitive ion sensors.

Method:

The peptide can be synthesized by the solid phase method disclosed byRovero, P. et al. (1991), Tetrahedron Lett., 32, 2639-2642 andcharacterized by ¹H-NMR spectroscopy, elemental analysis, and ion-spraymass spectrometry. Although a variety of conditions may be used in theself-assembly of cyclic peptide tubes, the following procedure hasprovided the most consistent results. Approximately 25 mg/ml suspensionof peptide subunit is clarified by the addition of 2.5 equivalents ofNaOH. The resulting peptide solution was centrifuged to remove traces ofsolid matter and then acidified by the addition of ⅓ volume of 1%trifluoroacetic acid in acetonitrile. Particles of cyclic peptide tubesgradually form as a white suspension over a period of hours cyclicpeptide tubes may then be collected by centrifugation and washedrepeatedly with distilled water to remove excess acids and salts. Forelectron microscopy and diffraction studies, a suspension of particlesof cyclic peptide tubes is sonicated briefly and small drops applied toglow discharged carbon support films on EM grids. Excess liquid isremoved by blotting and the grids frozen in liquid ethane slushaccording to the method disclosed by Adrian, M. et al., (1984) Nature308, 32-36 and Milligan, R. A. et al., (1984) Ultramicroscopy 13, 1-10.Grids are mounted in a Gatan cold stage and examined in a Philips CM12electron microscope operating at 120 kV. The specimen temperature was−175° C. during examination and imaging. Images are recorded at 35000×using strict low dose conditions at various defocus levels. For imageanalysis, micrographs are converted to optical density arrays using aPerkin-Elmer scanning microdensitometer with spot and step sizes equalto 2.86 Å at the specimen. Using the SUPRIM program package disclosed bySchroeter, J. P. et al. (1992), J. Structural Biology 109, 235-247, anumber of small areas from a single TEM image are rotationally andtranslationally aligned and then averaged.

SYNTHETIC METHODS

Synthesis of Linear Peptides:

The linear form of the target sequence may be synthesized according toconventional solid-phase methods and then cyclized in solution tofurnish the desired cyclic peptide subunit. A preferred method forsynthesizing linear peptides is provided as follows:

Step A: The C-terminal amino acid residue (aa.1) of the target linearpeptide is attached to a PAM resin (phenyl-acetamido-methyl).Hydroxymethyl PAM resin is a preferred PAM resin. Prior to use, it iswashed 4 times in DMF. A N-Boc-aa.1 (N-tert-butoxycarbonyl amino acid)is then linked to the washed PAM resin to form Boc-aa.1-PAM resin.Linkage is achieved by combining the PAM resin with 4 equivalents ofN-Boc-amino acid (D or L), 3.8 equivalents of HBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),and 6 equivalents of DIEA (N,N-diisopropylethylamine) in DMF. Theresultant mixture is then shaken for 1 hour. If the C-terminal aminoacid residue (aa.1) of the target linear peptide includes a potentiallyreactive side group, the side group is first blocked by conventionalblocking agent prior to its attachment to the PAM resin. After reactionis complete, the product PAM resin is washed 3 times in DMF for 1minute.

Step B: Because the product mixture will include a component ofunreacted PAM resin, the PAM resin then is capped by mixing it with 20equivalents of trimethylacetic anhydride and 10 equivalents of DIEA inDMF and shaking the resultant mixture overnight. The capped PAM resinbearing an N-Boc-amino acid residue is then washed 3 times in DMF and 3further times in CH₂Cl₂.

Step C: The protected amino group of the Boc-aa.1-PAM-resin is thendeprotected by treatment with neat TFA to form aa.1-PAM-resin. Step D:The deprotected Boc-aa.1-PAM-resin is then coupled to the second aminoacid residue (aa.2), i.e, the amino acid residue once removed from theC-terminus of the target linear peptide, to formBoc-aa.2-aa.1-PAM-resin. The second amino acid residue (aa.2) has achirality opposite the chirality of the C-terminal amino acid residue(aa.1), i.e., if aa.1 has a D chirality, aa.2 has an L chirality; ifaa.1 has an L chirality, aa.2 has an D chirality. The deprotectedBoc-amino acid-PAM-resin of step C is combined with 4 equivalents ofN-Boc-aa.2, 3.8 equivalents of HBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),and 6 equivalents of DIEA (N,N-diisopropylethylamine) in DMF. Thereaction mixture is then shaken for one hour.

Step E: The protected amino group of the Boc-aa.2-aa.1-PAM-resin is thendeprotected by treatment with neat TFA to produce aa.2-aa.1-PAM-resin.

Step F: Steps D and E are then repeated as required to couple the thirdand subsequent amino acid residues in succession to the nascent peptidechain to form a reaction product having the structure aa.n-aa(n-1)- . .. aa.1-PAM-resin. The chirality of the even amino acids is opposite thecharity of the odd amino acids.

Step G: After the synthesis of the target linear peptide is complete, itis cleaved from the PAM resin. Cleavage is achieved by treatment of thePAM-resin with a 10:1:0.5 mixture of HF, anisole, and dimethylsulfide at0° C. for 1 hour. The cleavage product may then be extracted from thereaction mixture with aqueous acetic acid (50% v/v) and lyophilized. Ifthe target linear peptide includes protected side groups, these sidegroups may be deprotected at this time. The product may then be verifiedby mass spectrometry.

Synthesis of Selectively N-Alkylated Linear Peptides:

The linear form of selectively N-alkylated target peptides may besynthesized according to a modification of conventional solid-phasemethods of peptide synthesis. The linear form of selectively N-alkylatedtarget peptides are then cyclized in solution to furnish the desiredselectively N-alkylated cyclic peptide. The method for synthesizinglinear form of selectively N-alkylated target peptides employsselectively N-alkylated N-Boc-amino acids. Preferred methods forsynthesizing these N-alkylated amino acids and selectively N-alkylatedlinear peptides are provided as follows:

N-Alkylated amino acids may be synthesized according to the method ofS.T. Cheung et al. (Canadian Journal of Chemistry (1977), vol. 55, p906; Canadian Journal of Chemistry (1977), vol. 55, p 911; and CanadianJournal of Chemistry (1977), vol. 55, p 916.) Briefly, 8 equivalents ofmethyl iodide were combined with tetrahydrofuran (THF) at 0° C. undernitrogen and stirred to form a suspension. Other alkyl iodides and alkylbromides may be substituted for the methyl iodide. To this suspensionwas added 1 equivalent of N-Boc-aa (N-tert-butoxycarbonyl amino acid) asa solid and 3 equivalents of sodium hydride. The resulting mixture wasthen stirred at room temperature under nitrogen for 24 hours. After 24hours, excess NaH was quenched by the careful addition of an H₂O to thereaction mixture. The mixture was then evaporated and the oily residuepartitioned between Et₂O and water. The Et2O layer was then washed withaqueous NaHCO₃. The combined aqueous extracts were then acidified to pH3 with aqueous citric acid (5%). The acidified product was thenextracted into EtOAc. The combined EtOAc layer was then serially washedwith H₂O, aqueous sodium thiosulfate, H₂O, and brine. The product wasthen dried over MgSO₄ and subsequently recrystallized. A typical yieldis 86%.

Selectively N-alkylated linear peptides may be synthesized by amodification of the method provided above for the synthesis ofnon-N-methylated linear peptides. N-Alkylated N-Boc-amino acids are lessreactive with respect to coupling reactions as compared tonon-N-alkylated N-Boc amino acids. As a consequence, coupling reactionsinvolving N-alkylated N-Boc-amino acids may be less efficient.Accordingly, in order to achieve a high over all yield, it is oftenuseful to follow up each coupling reaction with one or more recouplingreactions.

A selectively N-alkylated target peptide may be synthesized as follows:

Step A: The C-terminal amino acid residue (aa.1) of the target linearpeptide is attached to a PAM resin (phenyl-acetamido-methyl).Hydroxymethyl PAM resin is a preferred PAM resin. Prior to use, it iswashed 4 times in DMF. A N-Boc-aa.1 (N-tert-butoxycarbonyl amino acid)or a N-alkylated N-Boc-aa.1 is then linked to the washed PAM resin toform Boc-aa.1-PAM resin. Linkage is achieved by combining the PAM resinwith 4 equivalents of N-Boc-aa.1 (D or L) or N-alkylated N-Boc-aa.1 (Dor L), 3.8 equivalents of HBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),and 6 equivalents of DIEA (N,N-diisopropylethylamine) in DMF. Theresultant mixture is then shaken for 1 hour. If the C-terminal aminoacid residue (aa.1) of the target linear peptide includes a potentiallyreactive side group, the side group is first blocked by conventionalblocking agent prior to its attachment to the PAM resin. After reactionis complete, the product PAM resin is washed 3 times in DMF for 1minute.

Step B: Because the product mixture will include a component ofunreacted PAM resin, the PAM resin then is capped by mixing it with 20equivalents of trimethylacetic anhydride and 10 equivalents of DIEA inDMF and shaking the resultant mixture overnight. The capped PAM resinbearing an N-Boc-amino acid residue or N-alkylated N-Boc amino acid isthen washed 3 times in DMF and 3 further times in CH₂Cl₂.

Step C: The protected amino group of the Boc-aa.1-PAM-resin orN-alkylated N-Boc-aa.1-Pam resin is then deprotected by treatment withneat TFA to form aa.1-PAM-resin or N-alkylated aa.1-PAM resin,respectively.

Step D: The deprotected Boc-aa.1-PAM-resin or N-alkylated aa.1-PAM resinis then coupled to the second amino acid residue (aa.2 or N-alkyl aa.2),i.e, the amino acid residue once removed from the C-terminus of thetarget linear peptide, to form Boc-aa.2-aa.1-PAM-resin, Boc-aa.2-N-alkylaa.1-PAM-resin, N-alkyl Boc-aa.2-aa.1-PAM-resin, or N-alkylBoc-aa.2-N-alkyl aa.1-PAM-resin. The second amino acid residue (aa.2)has a chirality opposite the chirality of the C-terminal amino acidresidue (aa.1), i.e., if aa.1 has a D chirality, aa.2 has an Lchirality; if aa.1 has an L chirality, aa.2 has an D chirality. Thedeprotected Boc-aa.1-PAM-resin or N-alkyl Boc-aa.1-PAM-resin of step Cis combined with 4 equivalents of N-Boc-aa.2 or N-alkyl N-Boc-aa.2, 3.8equivalents of HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate), and 6 equivalents of DIEA(N,N-diisopropylethylamine) in DMF. The reaction mixture is then shakenfor one hour. If coupling is occurring with N-alkyl Boc-aa.1-PAM-resin,the efficiency of the initial coupling reaction may be relatively low.In this event, an aliquot of the reaction mixture may then be assayed bythe chloranil test. If the test is positive, the reaction product istreated second time with the above reactants to achieve an essentiallyquantitative yield of Boc-aa.2-N-alkyl aa.1-PAM-resin or N-alkylBoc-aa.2-N-alkyl aa.1-PAM-resin.

Step E: The protected amino group of the product of Step D, i.e.,Boc-aa.2-aa.1-PAM-resin, Boc-aa.2-N-alkyl aa.1-PAM-resin, N-alkylBoc-aa.2-aa.1-PAM-resin, or N-alkyl Boc-aa.2-N-alkyl aa.1-PAM-resin, isthen deprotected by treatment with neat TFA to produceaa.2-aa.1-PAM-resin, aa.2-N-alkyl aa.1-PAM-resin,N-alkyl-aa.2-aa.1-PAM-resin, or N-alkyl-aa.2-N-alkyl aa.1-PAM-resin.

Step F: Steps D and E are then repeated as required to couple the thirdand subsequent amino acid residues in succession to the nascent peptidechain to form a target selectively N-alkylated linear peptide linked toresin.

Step G: After the synthesis of the target selectively N-alkylated linearpeptide is complete, it is cleaved from the PAM resin. Cleavage isachieved by treatment of the PAM-resin with a 10:1:0.5 mixture of HF,anisole, and dimethylsulfide at 0° C. for 1 hour. The cleavage productmay then be extracted from the reaction mixture with aqueous acetic acid(50% v/v) and lyophilized. If the target selectively N-alkylated linearpeptide includes protected side groups, these side groups may bedeprotected at this time. The product may then be verified by massspectrometry.

Synthesis of Linear Peptide Precursors of Gated Cyclic Peptides:

The linear form of selectively N-substituted target peptides may besynthesized according to a modification of conventional solid-phasemethods of peptide synthesis. Gated cyclic peptides can be formed bycyclization of linear peptides selectively N-substituted with respect totheir peptide backbone amino groups. Preferred substitutions for forminggated cyclic peptides are illustrated in FIG. 7. Each of the preferredsubstitutions includes a heterocyclic structure linked via an alkylchain, viz. N—(CH₂)_(n)-heterocycle, where N is a peptide amino nitrogenand “n” lies between 1 and 5. The distal end of the alkyl chain isbonded to a selected peptide amino nitrogen on the peptide backbone. Inthe preferred embodiment, all N-substitutions are on the same face ofthe cyclic peptide. Preferred heterocyclic structure include imidazole,pyridine, 2,2′:6,2″terpyridine, and 2,2′-bipyridine.

N-substituted N-Boc-amino acids are employed for synthesizing the linearform of selectively N-substituted target peptides. The method of S. T.Cheung et al. (Canadian Journal of Chemistry (1977), vol. 55, p 906;Canadian Journal of Chemistry (1977), vol. 55, p 911; and CanadianJournal of Chemistry (1977), vol. 55, p 916.) may be employed forsynthesizing these N-substituted amino acids. The synthetic methodemploys a haloalkyl-heterocycle as a substrate, i.e,X—(CH₂)_(n)-heterocycle, where X is a halogen and “n” lies between 1 and5. Preferred halogens include bromine and iodine. Preferred alkyl groupsinclude (CH₂)_(n) were n lies between 1 and 5. The halogen is positionedat one end of the alkyl chain distal with respect to the attachment ofthe alkyl chain to the heterocycle.

Preferred haloalkyl-heterocyclic substrates may be obtained as follows:

-   4-(Bromomethyl)-1-H imidazole may be synthesized according to the    method of D. E. Ryono et al. in German Patent DE 3309014 (Sep. 29,    1983), claiming priority from U.S. patent application Ser. No.    356941 (Mar. 15, 1982) or according to the method of W. Schunack in    Arch. Pharm. (1974), vol. 307(1), pages 46-51.-   4-(2-Bromoethyl)-1-H imidazole may be synthesized according to the    method of E. T Chen in Anal. Chem. (1993), vol. 65(19), pages    2563-2567.-   4-(3-Bromopropyl)-1-H imidazole may be synthesized according to the    method of P. Franchetti et al. in Farmaco, Ed. Sci., vol. 29(4),    pages 309-316 and according to the method of W. M. P. B. Menge et    al. in J. Labelled Compd. Radiopharm. (1992), vol. 31(10), pages    781-786.-   3-(Bromomethyl)-pyridine may be synthesized according to the method    of R. Jokela et al. in Heterocycles 1985, vol. 23(7), pages 1707-22.-   3-(2-Bromoethyl)-pyridine may be synthesized according to the method    of A. Lochead et al. in European Patent Application No. EP 320362    (Jun. 14, 1989) and EP 88-403079 (Dec. 6, 1988), claiming priority    from French patent application FR 87-17044 or according to the    method of R. A. R. Bruneau et al in European Patent application EP    284174 (Sep. 28, 1988) and EP 88-300281 (Jan. 14, 1988), claiming    priority from EP 87-400122 (Jan. 19, 1987) and EP 87-401798 (Jul.    31, 1987).-   3-(3-Bromopropyl)-pyridine may be synthesized according to the    method of A. W. Van der Made et al. in Recl. Trav. Chim. Pays-Bas    (1990), vol. 109(11), pages 537-551.-   3-(4-Bromobutyl)-pyridine may be synthesized according to the method    of J. W. Tilley et al. in the Journal of Organic Chemistry (1987),    vol. 52(12), pages 2469-2474 or according to the method of U. R.    Patel in U.S. Pat. No. 4,855,430 (Aug. 8, 1989) or according to the    method of M. Carson et al. in U.S. Pat. No. 4,663,332 (May 5, 1987).-   3-(Iodomethyl)-pyridine may be synthesized according to the method    of G. G. Abashev in USSR Patent No. SU 1692985 A1 (Nov. 23, 1991).-   4′-(4-Bromobutyl)-2,2′:6,2″-terpyridine may be synthesized according    to the method of J. K. Bashkin in PCT International Patent    Application No. WO 9119730 A1 (Dec. 26, 1991) or WO 91-US3880 (Jun.    3, 1991).-   5-(Bromomethyl)-2,2′-bipyridine may be synthesized according to the    method of J. Uenishi et al. in the Journal of organic Chemistry    (1993), vol. 58(16), pages 4382-4388 or according to the method    of B. Imperiali et al. in the Journal of Organic Chemistry (1993),    vol. 56(6), pages 1613-1616.

Briefly, 8 equivalents of a haloalkyl-heterocyclic substrate, asindicated above, is combined with tetrahydrofuran (THF) at 0° C. undernitrogen and stirred to form a suspension. To this suspension is added 1equivalent of N-Boc-aa (N-tert-butoxycarbonyl amino acid) as a solid and3 equivalents of sodium hydride. The resulting mixture is then stirredat room temperature under nitrogen for 24 hours. After 24 hours, excessNaH is quenched by the careful addition of an H₂O to the reactionmixture. The mixture is then evaporated and the oily residue partitionedbetween Et₂O and water. The Et₂O layer is then washed with aqueousNaHCO₃. The combined aqueous extracts are then acidified to pH 3 withaqueous citric acid (5%). The acidified product is then extracted intoEtOAc. The combined EtOAc layer is then serially washed with H₂O,aqueous sodium thiosulfate, H₂O, and brine. The product is then driedover MgSO₄ and subsequently recrystallized.

Selectively N-substituted linear peptides may be synthesized accordingto the method provided above for the synthesis of N-alkylated orN-methylated linear peptides. If an N-substituent is bulky, theN-substituted N-Boc-amino acids can be even less reactive with respectto coupling reactions than N-methyl N-Boc amino acids due to sterichinderance. As a consequence, coupling reactions involving N-substitutedN-Boc-amino acids may be slow and relatively inefficient. Accordingly,in order to achieve a high over all yield, it is often useful to followup each coupling reaction with repeated recoupling reactions.

A selectively N-substituted target peptide may be synthesized asfollows:

Step A: The C-terminal amino acid residue (aa.1) of the target linearpeptide is attached to a PAM resin (phenyl-acetamido-methyl).Hydroxymethyl PAM resin is a preferred PAM resin. Prior to use, it iswashed 4 times in DMF. A N-Boc-aa.1 (N-tert-butoxycarbonyl amino acid)or a N-substituted N-Boc-aa.1 is then linked to the washed PAM resin toform Boc-aa.1-PAM resin. Linkage is achieved by combining the PAM resinwith 4 equivalents of N-Boc-aa.1 (D or L) or N-substituted N-Boc-aa.1 (Dor L), 3.8 equivalents of HBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),and 6 equivalents of DIEA (N,N-diisopropylethylamine) in DMF. Theresultant mixture is then shaken for 1 hour. If the C-terminal aminoacid residue (aa.1) of the target linear peptide includes a potentiallyreactive side group, the side group is first blocked by conventionalblocking agent prior to its attachment to the PAM resin. After reactionis complete, the product PAM resin is washed 3 times in DMF for 1minute.

Step B: Because the product mixture will include a component ofunreacted PAM resin, the PAM resin then is capped by mixing it with 20equivalents of trimethylacetic anhydride and 10 equivalents of DIEA inDMF and shaking the resultant mixture overnight. The capped PAM resinbearing an N-Boc-amino acid residue or N-substituted N-Boc amino acid isthen washed 3 times in DMF and 3 further times in CH₂Cl₂.

Step C: The protected amino group of the Boc-aa.1-PAM-resin orN-substituted N-Boc-aa.1-Pam resin is then deprotected by treatment withneat TFA to form aa.1-PAM-resin or N-substituted aa.1-PAM resin,respectively.

Step D: The deprotected Boc-aa.1-PAM-resin or N-substituted aa.1-PAMresin is then coupled to the second amino acid residue (aa.2 or N-alkylaa.2), i.e, the amino acid residue once removed from the C-terminus ofthe target linear peptide, to form Boc-aa.2-aa.1-PAM-resin,Boc-aa.2-N-alkyl aa.1-PAM-resin, N-alkyl Boc-aa.2-aa.1-PAM-resin, orN-alkyl Boc-aa.2-N-alkyl aa.1-PAM-resin. The second amino acid residue(aa.2) has a chirality opposite the chirality of the C-terminal aminoacid residue (aa.1), i.e., if aa.1 has a D chirality, aa.2 has an Lchirality; if aa.1 has an L chirality, aa.2 has an D chirality. Thedeprotected Boc-aa.1-PAM-resin or N-alkyl Boc-aa.1-PAM-resin of step Cis combined with 4 equivalents of N-Boc-aa.2 or N-alkyl N-Boc-aa.2, 3.8equivalents of HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate), and 6 equivalents of DIEA(N,N-diisopropylethylamine) in DMF. The reaction mixture is then shakenfor one hour. If coupling is occurring with N-alkyl Boc-aa.1-PAM-resin,the efficiency of the initial coupling reaction may be relatively low.In this event, an aliquot of the reaction mixture may then be assayed bythe chloranil test. If the test is positive, the reaction product istreated second time with the above reactants to achieve an essentiallyquantitative yield of Boc-aa.2-N-alkyl aa.1-PAM-resin or N-alkylBoc-aa.2-N-alkyl aa.1-PAM-resin.

Step E: The protected amino group of the product of Step D, i.e.,Boc-aa.2-aa.1-PAM-resin, Boc-aa.2-N-alkyl aa.1-PAM-resin, N-alkylBoc-aa.2-aa.1-PAM-resin, or N-alkyl Boc-aa.2-N-alkyl aa.1-PAM-resin, isthen deprotected by treatment with neat TFA to produceaa.2-aa.1-PAM-resin, aa.2-N-alkyl aa.1-PAM-resin,N-alkyl-aa.2-aa.1-PAM-resin, or N-alkyl-aa.2-N-alkyl aa.1-PAM-resin.

Step F: Steps D and E are then repeated as required to couple the thirdand subsequent amino acid residues in succession to the nascent peptidechain to form a target selectively N-substituted linear peptide linkedto resin.

Step G: After the synthesis of the target selectively N-substitutedlinear peptide is complete, it is cleaved from the PAM resin. Cleavageis achieved by treatment of the PAM-resin with a 10:1:0.5 mixture of HF,anisole, and dimethylsulfide at 0° C. for 1 hour. The cleavage productmay then be extracted from the reaction mixture with aqueous acetic acid(50% v/v) and lyophilized. If the target selectively N-substitutedlinear peptide includes protected side groups, these side groups may bedeprotected at this time. The product may then be verified by massspectrometry.

Cyclization of Linear Peptides:

The target linear peptides, selectively N-alkylated target linearpeptides, and selectively N-substituted target linear peptides, whosesyntheses are described above, may each be cyclized according to thefollowing protocol:

A solution of the linear peptide in DMF (1 mM) is treated with TBTU(2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate,3 mM), HOBt (1-Hydroxybenzotriazole, 3 mM) and DIEA(diisopropylethylamine, 1% v/v) at 5° C. for 12 hours to give thedesired cyclic peptide monomer. The product may be purified byreverse-phase HPLC purification. A typical yield for the cyclization ofN-methylated linear peptides octomer is 70% yield.

Alternative Method for Peptide Synthesis and Cyclization:

Alternatively, peptides containing an Asp residue can be synthesized andcyclized by the solid phase method disclosed by Rovero, P. et al.(1991), Tetrahedron Lett., 32, 2639-2642. Briefly, Boc-Asp(N-tert-butoxycarbonyl aspartic acid) is linked to PAM resin(phenyl-acetamido-methyl) through the β-carboxylic function while theα-carboxylic groups is protected as a fluorenylmethyl ester (OFm).Boc-Asp(β-PAM-resin)OFm may be purchased from Bachem A G, Switzerland. Alinear peptide having the D-L chirality motif may then be built upon theBoc-Asp(β-PAM-resin)OFm according to the classical Boc/Benzyl strategyusing an automatic or semi-automatic peptide synthesizer, e.g., LabortecSP 640. Synthesis is achieved by consecutively adding Boc-protectedamino acids according to the BOP coupling procedure, i.e., 3 equivalentsBoc-amino acid, 3 equivalent BOP and 6 equivalent DIEA in DMF for 1hour. Completeness may be achieved by repeating each coupling twice.Once the synthesis of the linear peptide is complete, it ready forcyclization. Prior to cyclization, the N-terminal amino group wasdeprotected with TFA and the C-terminal fluorenylmethyl ester wasdeblocked with piperidine (20% v/v piperidine in DMF) for 3+7; minutes.Cyclization was then achieved by treatment with 3 equivalents of BOP(benzotriazolyl-N-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate) and 6 equivalents of DIEA(N,N-diisopropylethylamine) in DMF for 3 hours. If the cyclizationreaction is incomplete, the BOP treatment may be repeated. Deprotectionof the side chains and cleavage of the cyclic peptide from the resin maybe achieved by treatment with a 10:1:0.5 mixture of HF, anisole, anddimethylsulfide at 0° C. for 1 hour. The product may then be extractedfrom the reaction mixture with aqueous acetic acid (50% v/v) andlyophilized.

Capped and gated cyclic peptides may also be synthesized according tothe above method by cyclizing the corresponding N-substituted linearpeptides.

1. A method for constructing molecular tubes comprising the followingsteps: Step A: providing a first solution having charged cyclic peptidessolubilized therein, each of said cyclic peptides having one or moreionized amino acid residues for imparting a net charge to said chargedcyclic peptides, said charged cyclic peptides being homodetic and havingan amino acid sequence with a length between 6 and 16 amino acidresidues, a repeating D-L chirality motif, and an even number of aminoacid residues, wherein all of said ionized amino acid residues areeither ionized acidic amino acid residues or ionized basic amino acidresidues; and then Step B: neutralizing the charge of the charged cyclicpeptides of said Step A by changing the pH of the first solution forforming a second solution having neutral cyclic peptides lacking a netcharge and for promoting hydrogen bond formation; and then Step C:forming a suspension of molecular tubes within said second solution byallowing self-assembly of the neutral cyclic peptides with stacking ofthe neutral cyclic peptides upon one another in an anti-parallel fashionwith a formation of β-sheet hydrogen bonds between adjacent neutralcyclic peptides.
 2. A molecular tube comprising: a plurality of cyclicpeptides, each of said peptides being homodetic and having an amino acidsequence with a length between 6 and 16 amino acid residues, a repeatingD-L chirality motif, and an even number of amino acid residues; each ofsaid cyclic peptides including an ionizable amino acid residue; saidmolecular tube being formed in solution by self-assembly induced byneutralization of the ionizable amino acid residue and stacking of saidcyclic peptides upon one another in an anti-parallel fashion with aformation of β-sheet hydrogen bonds between adjacent cyclic peptides. 3.A molecular tube as described in claim 2 further comprising: a terminalcyclic peptide having a ring structure with a first face and a secondface, the first face having hydrogen bond donors, the second face beingdevoid of hydrogen bond donors; said terminal cyclic peptide forming aterminal end of said molecular tube by stacking the first face thereontoin an anti-parallel fashion with a formation of β-sheet hydrogen bonds.4. A molecular tube as described in claim 3 wherein the second face ofsaid terminal cyclic peptide has alkyl-block amino groups thereon.
 5. Amethod as described in claim 1 wherein: in said Step A, all of saidionized amino acid residues are ionized acidic amino acid residues andimpart a net negative charge to said charged cyclic peptides; and insaid Step B, the net negative charge of said charged cyclic peptides ofsaid Step A is neutralized by protonation by decreasing the pH of thefirst solution.
 6. A method as described in claim 1 wherein: in saidStep A, all of said ionized amino acid residues are ionized basic aminoacid residues and impart a net positive charge to said charged cyclicpeptides; and in said Step B, the net positive charge of said chargedcyclic peptides of said Step A is neutralized by deprotonation byincreasing the pH of the first solution.